anti-D Reagents Research Articles

Contingency Plan for Anti-D Reagent Shortages for RhD Testing: Validation of Using Diluted Anti-D Reagents

Contingency Plan for Anti-D Reagent Shortages for RhD Testing: Validation of Using Diluted Anti-D Reagents

Application of RhD Blood Group to Simulate Antibody Identification Test in Immunohematology Education.

Immunohematology skill education is an important part of the transfusion medicine professional training. We tried to solve the difficulty of obtaining suitable and sufficient positive samples in the immunohematology education. Different identification panels and panel cells were created by RhD-positive red blood cells (RBCs) and RhD-negative RBCs, according to the underlying antibodies. Diluted anti-D reagent was used as simulated plasma for identification. The antibody identification of single antibody with dose-effect and two antibodies present at the same time were successfully simulated. It is a practical and cheap method for antibody identification training to use RhD blood group, especially when positive samples are short.

RHD Genotyping to Resolve Weak and Discrepant RHD Phenotypes: The “Serenissima” Experience

Background: A considerable number of RHD alleles responsible for weak and partial D phenotypes have been identified. Serological determination of these phenotypes is often doubtful and makes genetic analysis of RHD gene highly desirable in transfusion recipients and pregnant women. Aim: We report the experience of Mestre Blood Bank in analysis of the RHD gene in six years from 2018 to 2023. Methods: Subjects for RHD gene analysis were selected for presence of a serological weak D phenotype, defined as reactivity of RBCs with an anti-D reagent giving no or weak (≤2+) score in initial testing but agglutinating moderately or strongly with anti human globulin (AHG). These samples were selected for genotyping using the microarray-based method Bead-Chip supplied by Werfen. Results: From 2018 to 2023, we selected, for RHD gene analysis, 555 subject with D weak phenotype; 86 subjects (15.5%) were D positive and 56 (10.1%) were D negative, without variant, in 413 subjects a D weak or a D variant was observed. Discussion: Many serological weak D phenotypes are associated to RHD gene mutations leading to one or more amino acids substitutions in the RhD protein predicted to be within or below the RBC membrane, causing decreased antigen expression on the red cell surface. Prevalence of serological weak D phenotypes varies by race and ethnicity. Serological weak D phenotypes are the most common D variants detected in Caucasians (0.2%-1.0%). The majority, as in our series, are associated with weak D type 1, 2 or 3. Our data confirmed a high prevalence of weak D type 1 and type 2, but we observed a high prevalence of type 11 and 15 and of the uncommon type 18 too. The most common partial D phenotypes in Europe are DNB, DVI, and DVII. Our data confirmed a high prevalence of D partial type VI. Studies indicate that D partial transfusion recipients are at risk of forming alloanti-D when exposed to conventional RhD-positive blood units.

Frequency of the 'Asia type' DEL with weak D phenotype in Chinese.

DEL individuals account for 9-30% of serological RhD negative population in east Asia and majority of them carrying the RHD*DEL1 allele are referred to as 'Asia type' DEL individuals. There is a lack of data on the molecular basis for 'Asia type' DELs with weak RhD phenotype. Therefore, the aim of this study is to unveil 'Asia type' DELs by elucidating the genetic background and analyzing the serological results METHODS: With a microplate typing protocol, RhD characterization was performed in samples from one million blood donors collected at Chengdu blood center during the period from 2019 to 2022. RhD confirmatory test was performed by direct antiglobulin test and indirect antiglobulin test with five anti-D reagents to detect RhD variants. Molecular characterization of samples categorized as RhD variants was studied by using direct genomic DNA sequencing and RHD zygosity analysis, followed by adsorption and elution tests conduced for samples carrying RHD*DEL1 allele to confirm the presence of RhD antigens on the red cells RESULTS: We reported here 21 RhD variant samples were detected by micro-column gel agglutination assay with IgG anti-D antibodies. Moreover, the agglutination reaction was stronger with IgG anti-D reagents in micro-column gel card than with IgM/IgG blended anti-D antibodies. Each of the 21 samples carried the RHD*DEL1 allele, which indicated that they were 'Asia type' DEL. Of the 21 'Asia type' DEL samples, 9 samples were detected to be RHD+/ RHD+ homozygotes, whereas the other 12 samples were RHD+/RHD- hemizygotes. Among the samples phenotyped for RhCE, seven were CCee and four were Ccee. In this study, DEL samples carrying RHD*DEL1 showed weak RhD phenotype with some anti-D reagents in RhD confirmatory test, which suggest that a serology strategy using several anti-D reagents may be helpful to detect this 'Asia type' DEL. Further studies are needed to elucidate whether the 'Asia type' DELs with weak RhD phenotype have stronger antigenicity and could cause serious transfusion reaction.

A Korean family with RHD*DNT only detectable after anti-D alloimmunization.

Identification of DNT, a rare partial D, can be challenging, as it is difficult to distinguish from D+. This study aimed to identify DNT individuals by analyzing the DNT proband's family members, characterize DNT, and propose management strategies. Family members of the first Korean DNT proband were recruited. RHD genotyping was conducted, and weak D tests were carried out using several anti-D reagents. Three DNT individuals were identified among 6 family members, including 1 with an anti-D alloantibody. As DNT red cells exhibited strong reactivity with all anti-D clones, DNT was serologically indistinguishable from D+. Moreover, unusual serologic findings in DNT individuals only became apparent after anti-D alloimmunization. We recommend DNT individuals as candidates for Rh immune globulin prophylaxis during the perinatal period and transfusions with D- blood components. An anticipatory RHD genotyping is suggested for partial D family members to prevent potential partial D individuals from becoming alloimmunized.

Serological and molecular characterisation of the most prevalent weak D variants in Croatian population.

Existence of hundreds of RHD gene variants contributes to variable D antigen expression and inconsistencies in reporting the RHD results. The aim of the study was to determine the serological and molecular characteristics of the most prevalent RHD alleles encoding serologically weak D variants. Blood donors (n=145 924) were typed for D antigen using the direct serologic micromethod. Nonreactive samples were analysed in IAT method with the IgM/IgG anti-D monoclonal blend, and 0,2% (n=263) confirmed weak D antigen expression. After genomic DNA extraction (Qiaqen, Germany), RHD genotyping was performed using in house reagents and PCR-SSP kits (Inno-Train, Germany). The prevalence of serologically weak D in blood donor population was 0.2% (n=263). RHD genotyping confirmed weak D allele in 92.4% and partial D allele in 7.6%. The most common was weak D type 1 (49.7%) followed by weak D type 3 (24.7%) and type 2 (9.5%). Relatively high frequency was detected for weak D type 14 (4.6%) and type 64 (2.3%). In the category of partial D phenotypes, only DVI variant was found. Direct typing has shown great variability in the strength of reactions with different clones of anti-D reagents. Weak D type 1 is the most common weak D variant in Croatian blood donor population. The frequency of D variants and distribution of Rh phenotypes in our study was in concordance with other studies. It has been shown that serological methods and the combination of clones used, cannot distinguish variant D types, which justifies the use of molecular methods.

Transfusion support for a woman with RHD*09.01.02 and the novel RHD*01W.161 allele in trans.

According to recent work group recommendations, individuals with the serologic weak D phenotypes should be RHD genotyped and individuals with molecular weak D types 1, 2, 3, 4.0, or 4.1 should be treated as D+. We report an African American woman with a long-standing history of metrorrhagia, who presented for infertility evaluation. Blood grouping showed AB with a possible subgroup of A, based on mixed-field agglutination, and a serologic weak D phenotype. Results from routine red cell genotyping for the RHD gene was incongruent with the serologic RhCE phenotype. For the surgical procedure, the patient was hence scheduled to receive group AB, D- RBC transfusions. Subsequent molecular analysis identified the ABO*A2.01 and ABO*B.01 alleles for the ABO genotype and the novel RHD allele [NG_007494.1(RHD):c.611T>A] along with an RHD*09.01.02 allele for the RHD genotype. Using a panel of monoclonal anti-D reagents, we showed the novel RHD(I204K) allele to represent a serologic weak D phenotype, despite occurring as a compound heterozygote, designated RHD*weak D type 161 (RHD*01W.161). Individuals with a weak D type 4.2 allele are prone to anti-D immunization, while the immunization potential of novel RHD alleles is difficult to predict. For now, patients should be treated as D- in transfusion and pregnancy management, when they harbor a novel RHD allele along with any weak D allele other than weak D types 1, 2, 3, 4.0, or 4.1. This study exemplifies strategies for how and when a laboratory should proceed from routine genotyping to nucleotide sequencing before any decisions on transfusion practice is made.

Detection of Rh Antibodies in Patient Plasma Using Genome Engineered Induced Pluripotent Stem Cell-Derived Red Cells

Background: Despite transfusion of Rh matched red cells for patients with sickle cell disease, Rh alloimmunization remains a persistent challenge. Rh specificities can be complex, resulting from RH genetic diversity found in patients and donors. Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens. We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to both screen for Rh alloimmunization and to aid complex antibody identification.Methods: We generated a panel of iPSCs that include Rh null, D–, lack the high prevalence antigens hr S or hr B, or express uncommon Rh antigens such as V, VS, Go a, or DAK. For the Rh null line, we used CRISPR/Cas9 genetic engineering to disrupt RHCE via a large deletion in a D- iPSC. For D–, RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using zinc finger nucleases resulting in a line that constitutively expresses RhD but no RhCE. iPSCs with uncommon variants were reprogrammed from RH genotyped donors or engineered similar to the generation of the D– line. Hematopoietic differentiation by embryoid body formation was used to generate hematopoietic progenitors that were subsequently cultured towards the erythroid lineage. Mature iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination assays.Results: Rh null iPSC-derived RBCs (iRBCs) showed complete absence of cell surface Rh protein by flow cytometry, while D– iRBCs showed Rh protein expression levels comparable to D-ce+ iRBCs using an anti-D/CE antibody. We assessed RBC agglutination of Rh null, D–, hr S-, hr B-, VVS+, Go a+, and DAK+ iRBCs using standard Rh typing reagents (Ortho). The reprogrammed uncommon donor iRBCs agglutinated with monoclonal anti-Rh antibodies as predicted by RH genotype, while the Rh null iRBCs showed no agglutination with all 5 common Rh antibodies and D– iRBCs showed agglutination with anti-D reagents only. Rh null iRBCs showed no agglutination against patient plasma containing anti-D, while D– iRBCs agglutinated. While D- RHCE*ce homozygous iRBCs showed strong agglutination against patient plasma containing anti-hr S, Rh null, D–, and hr S- iRBCs did not agglutinate. No iRBCs showed agglutination by plasma containing anti-V/VS while VVS+ iRBCs showed strong agglutination. Similarly, no iRBCs showed agglutination by plasma containing anti-Go a while Go a+ iRBCs showed strong agglutination. Detection of most antibodies against Rhce on iRBCs was enhanced by ficin treatment whereas antibodies with D specificity did not require ficin treated cells for detection.Conclusion: We suggest that genetically engineered iPSCs expressing uncommon Rh antigen phenotypes that are difficult or impossible to obtain from red cell donors can expedite antibody identification. Rh null and D– iRBCs could be useful to discriminate antibodies against RhD versus RhCE. Customized iPSCs that lack high prevalence or express low prevalence Rh antigens could potentially standardize antibody evaluation in patients with complex Rh specificities. DisclosuresNo relevant conflicts of interest to declare.

A retrospective study on the prevalence of weak D antigen (Du) in a blood bank in a tertiary care hospital in Maharashtra, India

Background: One of the most immunogenic blood groups known to humans is the Rhesus (Rh) blood group. The Rh blood group with variable expression of D antigen is one of the complex systems in immunohematology. Sometimes, the variable expression of RhD antigen leads to the presence of weaker forms that are not detected by routine laboratory procedures. Aims: The study was conducted to determine the frequency of RhD negativity and weak D antigens. Settings and Design: This retrospective study was conducted in the Department of Immunohematology and Transfusion Medicine at a tertiary care center hospital for three years from June 2017 to June 2020. Materials and Methods: All the serial Rh grouping tests were conducted by the gel technique or by the tube method with two Anti-D reagents. Negative D antigen samples were further tested for weak D antigen by Coombs Test. Statistical analysis used the Statistical Package for Social Sciences (SPSS). Results: Out of a total of 17262 samples, 15400 (89.2%) tested Rh-positive, and 1866 (10.8%) tested Rh-negative. Weak D was positive in 52(0.027%) samples out of the RhD negative samples. Conclusion: The frequency of weak D antigen is low, but the strong immunogenicity of RhD antigen calls for the need for appropriate testing. This is of concern in RhD negative pregnant females, as it can cause alloimmunization if accidentally weak D antigen-positive blood is transfused.

Molecular Characteristics of the Serological Weak D Phenotype in Koreans.

Serological weak D is a reaction of 2+ or less to anti-D reagent and includes weak D and partial D phenotypes. Although identifying the RhD subtype is important for transfusion safety, serological tests are insufficient for defining the RhD subtype, and molecular tests are needed. To analyze the molecular characteristics of D variants in Koreans to facilitate the formulation of individualized transfusion strategies, molecular tests such as RhD genotyping using real-time polymerase chain reaction (PCR) and partial-D and/or weak-D sequence-specific amplification (SSP) were performed on 105 Korean Rare Blood Program (KRBP) patients exhibiting serological weak D. In total, 58 out of 68 serologically determined weak D KRBP patients were typed as having weak D or partial D phenotypes via RhD genotyping. In detail, eight (13.8%) were typed as partial DVa or DBS, nine (15.5%) as weak D type 15, and four others (6.8%) as partial DVI, partial DVII, weak D type 2, or weak D type 41 or 45, whereas the rest (n = 37, 63.8%) was typed as having either weak D or partial D. This suggests that serological weak D Koreans who require transfusion should be treated as D-negative.

Molecular characterization of RhD variant phenotypes among blood donors: A study from the coastal region of India

BackgroundRhD expression varies with population and ethnicity. Accurate typing of RhD antigen among blood donors is important to prevent development of anti-D among recipients of blood transfusion. We aimed to screen blood donors for variant D phenotypes and accurately characterize them by genotyping. Material and methodsWe have done prospective study on blood donors by performing RhD typing using three different commercial monoclonal anti-D reagents by both column agglutination and conventional tube techniques. Samples that showed ambiguous results were further screened with the Bio-Rad Partial RhD typing kit. Minor phenotyping for C, c, E, e antigens was performed. Multiplex PCR and Sequencing of all RHD exons with Sanger’s sequencing was performed for molecular characterization of variant D. ResultsA total of 16,974 blood donors were screened during the study period. Among them, 31 (0.18 %) donors were found to have a RhD variant phenotype. The male to female ratio was 10:1. The presence of ‘C’ antigen was noted among all RhD variant samples. Serological typing identified two samples as DV phenotype and the rest could not be characterized. Molecular genotyping characterized 90.3 % of the samples as Indian specific weak D type 150 variants. Three samples were subjected to Sangers sequencing and showed wild type pattern. ConclusionThe present study showed that the most common variant in this population was Weak D type 150. This study highlights that serological methods may serve as a screening tool, however, molecular techniques are essential for characterization of RhD variants.

Donation testing and transfusion transmissible infections

Donation testing and transfusion transmissible infections

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

BackgroundWe evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors.MethodsWe selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays.ResultsThe RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested.ConclusionOur results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Does transfusion of Asian-type DEL red blood cells to D- recipients cause D alloimmunization?

In 2005, the late George Garratty asked readers of TRANSFUSION, “Do we need to be more concerned about weak D antigens?” His question was prompted by his observation that “… there have been increasing numbers of reports of patients who had been transfused with donor blood having ‘weak D’ or undetectable D (e.g., DEL) who made anti-D.” Garratty reviewed the complexity of the D antigen and its variants, case reports of anti-D after transfusion of weak D RBCs to D- recipients, and variable D typing results depending on selection of anti-D reagents. He noted that most available data were from Europe and concluded that “countries with larger Asian or African populations … need to relate to statistics in their own countries.” Garratty’s question can be answered only when data are available for the many different scenarios in which different categories of weak D RBCs have been transfused to D- recipients. In this commentary, we address one of these scenarios, namely, the potential risk of D alloimmunization when Asian-type DEL (RHD*DEL1) RBCs are transfused to D- recipients in the United States. We suggest how this issue can be resolved by a focused study in pertinent communities in the United States.

Frequencies of ABO and Rhesus (D) blood group phenotypes among pregnant women attending antenatal clinic at Murtala Muhammad Specialist Hospital, Kano, Nigeria

Background: Pregnancy, though a physiological phenomenon, is sometimes associated with complications requiring blood transfusion. Effective and safe blood transfusion services require a working database of the frequencies and distribution of ABO and Rhesus (Rh) (D) blood groups. Aim: The aim of this study was to determine the frequencies and distribution of ABO and Rh (D) blood groups among pregnant women attending antenatal clinic at Murtala Muhammad Specialist Hospital, Kano, Nigeria. Materials and Methods: A total of 210 pregnant women who presented to the booking section of the clinic were recruited for the study. Sociodemographic information of the patients was obtained using a semistructured interviewer-administered questionnaire. ABO and Rh (D) blood groups were determined by the agglutination method using potent monoclonal anti-A, anti-B, and anti-D reagents. Results: Blood groups O, B, A, and AB accounted for 98 (46.7%), 53 (25.2%), 45 (21.4%), and 14 (6.7%), respectively. Rh (D) positivity was 202 (96.2%) whereas Rh (D) negativity was 8 (3.8%). Conclusion: ABO phenotype O blood group is the most common among pregnant women in this environment with the majority being Rh (D) positive.

Relationship between ABO blood group phenotypes and hypertensive disorders of pregnancy: A hospital-based cross-sectional study in Kano, North-West Nigeria

Background: Hypertensive disorders of pregnancy (HDP) complicate an estimated 5%–10% of pregnancies globally. Studies have linked non-O blood group with the development of HDP, especially preeclampsia (PE). However, these reports have not been consistent. This study aimed to determine the relationship between ABO blood group and HDP among pregnant women attending antenatal clinic at Murtala Muhammad Specialist Hospital, Kano, Nigeria. Materials and Methods: Two hundred and ten pregnant women with clinically diagnosed HDP and an equal number of age-matched normotensive controls were recruited for the study. Blood pressure was measured using a mercury sphygmomanometer and a Littman's stethoscope which was positioned on the medial aspect of the right arm at sitting position. Urinalysis was performed using a urine dipstick (Medi-Test Combi 9®). ABO blood groups were determined by tile agglutination method using potent monoclonal anti-A, anti-B, and anti-D reagents (Plasmatec Lab., Bridport, UK). Data were analyzed using Statistical Package for Social Sciences version 23.0. Chi-square test of association, Student's t-test, and logistic regression were used as statistical tools, and results were presented as frequencies, percentages, mean ± standard, odds ratio (OR), and confidence interval (CI); P ≤ 0.05 was considered statistically significant. Results: The mean age of the cases and controls was 26.20 ± 6.96 and 25.90 ± 6.37, P = 0.65, respectively. The mean gestational age of the cases and controls was 32.30 ± 4.15 and 29.06 ± 4.25, P = 0.01, respectively. The mean systolic and diastolic blood pressure of the cases and controls in mmHg were 176.62 ± 32.62 and 123.08 ± 6.40, P = 0.01, and 114.20 ± 20.08 and 76.43 ± 5.07, P = 0.01, respectively. Of the 210 individuals in the HDP group (cases), 90 (42.86%) had gestational hypertension (GH), 50 (23.81%) had PE, and 70 (33.33%) had eclampsia. Type O blood group was the most common group in both cases (93 [44.3%]) and controls (98 [46.7%]). This was followed by groups A (40 [19.0%] and 45 [21.45]), B (58 [27.6%] and 53 [25.2%]), and AB (19 [9.0%] and 14 [6.7%]) for cases and controls, respectively. There was no statistically significant difference in the frequencies of ABO blood group between the cases and controls (χ2 = 1.41, P = 0.70, df = 3). Compared to type O blood group individuals, non-O blood group individuals had 1.106 times odd of developing HDP than the controls (OR: 1.106, 95% CI: 0.753–1.625, P = 0.61). Similarly, types A, B, and AB blood group individuals had 1.292, 1.492, and 1.882 times odds of developing GH compared to blood Group O and controls (OR: 1.292, 95% CI: 0.670–2.490, P = 0.45; OR: 1.492, 95% CI: 0.813–2.740, P = 0.20; and OR: 1.882, 95% CI: 0.746–4.748, P = 0.18), respectively. Equally, types A, B, and AB blood group individuals had 1.243, 0.990, and 1.699 times odds of developing PE compared to blood group O individuals and controls (OR: 1.243, 95% CI: 0.561–2.754, P = 0.59; OR; 0.990, 95% CI: 0.442–2.217, P = 0.98; and OR: 1.699, 95% CI: 0.546–5.281, P = 0.36), respectively. Compared to type O blood group individuals, types A, B, and AB blood group individuals had 0.463, 0.690, and 0.901 times odds of developing eclampsia compared to controls (OR: 0.463, 95% CI: 0.199–1.079, P = 0.74; OR: 0.960, 95% CI: 0.505–1.825, P = 0.90; and OR; 0.901, 95% CI: 0.301–2.695, P = 0.85), respectively. Primiparity (OR: 2.320, 95% CI: 1.451 – 3.711, P = 0.01, for HDP) and gestational age (OR: 1.078, 95% CI: 1.013 – 1.148, P = 0.02 – gestational hypertension; OR: 1.328, 95% CI: 1.212 – 1.456, P = 0.01 – preeclampsia; OR: 1.467, 95% CI: 1.333 – 1.614, P = 0.01 – eclampsia) were identified as pregnancy related risk factors for HDP. Conclusion: The frequencies of ABO phenotypes in women with HDP are similar to that of normotensive pregnant women. There is no relationship between ABO phenotypes and HDP as observed in this study. However, nulliparity and gestational age were identified as risk factors for HDP. Similar hospital-based and population-based studies should be conducted to further evaluate this phenomenon in this environment.

The synonymous nucleotide substitution RHD 1056C>G alters mRNA splicing associated with serologically weak D phenotype.

D antigen is one of the most clinically significant blood group antigens. Variation of the RHD gene can cause weak D or partial D phenotypes. While most variations are missense substitutions with amino acid changes, those without are called "silent" or "synonymous" substitutions. Synonymous substitutions often have little effect on the protein, not altering the phenotype. However, effect on splicing can affect end-product protein. We report a new synonymous variation, RHD 1056C>G, that resulted in weak D phenotype, and predicted its effect with various in silico methods. Serologic testing of the D antigen with full sequencing of the RHD gene was done. Human Splice Finder was used to predict the effect of this variation, and validation of this method was done with all known RHD variations reported in the literature. RHD 1056C>G was predicted to cause the formation of an exonic splicing silencer (ESS) site. The creation of new ESS site potentially inhibits the splicing event, resulting alteration of splicing. This is similar to remodeling of splice acceptor or donor site, as this kind of deep exonic variation could affect the D antigen's quality or quantity. This is in concordance with serologic results, which showed only delayed weak agglutination to anti-D reagents. The analytic methods we applied showed good correlation with the actual phenotype, along with concordant results when analyzing other known variants reported in the literature. We conclude that RHD 1056C>G results in serologic weak D phenotype.

Anti-D reagents should be chosen accordingly to the prevalence of D variants in the obstetric population.

Resolving ambiguous results of D antigen typing is crucial for appropriate and rational administration of anti-D immunoprophylaxis and transfusion practice in obstetric population. The aim of the study was to establish selection criteria of anti-D reagents for our population. A total of 12689 samples from primiparous women in Split-Dalmatia County, Croatia, were typed for RhD antigen during the period of 5years. Ambiguous results were submitted to additional serologic investigation and genotyping. RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D ® and Ready gene CDE, Inno-Train, Kronberg, Germany). Relative frequencies and accompanying 95% confidence intervals were used to estimate the prevalence of variants. The prevalence of D variants was 0.42% (95% CI 0.31; 0.53). The most common partial D variant was D Va (RHD*05.05), with the prevalence of 0.08% (95% CI 0.03; 0.13). All weak D variants were weak D types 1, 2 and 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3). Weak D samples were distinguishable from partial D in routine typing due to the difference in reactivity of partial D samples with clones D7B8 and RUM-1. Cell line RUM-1 gives weak or negative reactions with partial DVa category. The most common partial D variant in our population is DVa. It is recommended to use cell lines which do not strongly agglutinate DVa variant in routine RhD typing. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required.

Serological weak D phenotypes: a review and guidance for interpreting the RhD blood type using the RHD genotype.

Approximately 0·2-1% of routine RhD blood typings result in a "serological weak D phenotype." For more than 50years, serological weak D phenotypes have been managed by policies to protect RhD-negative women of child-bearing potential from exposure to weak D antigens. Typically, blood donors with a serological weak D phenotype have been managed as RhD-positive, in contrast to transfusion recipients and pregnant women, who have been managed as RhD-negative. Most serological weak D phenotypes in Caucasians express molecularly defined weak D types 1, 2 or 3 and can be managed safely as RhD-positive, eliminating unnecessary injections of Rh immune globulin and conserving limited supplies of RhD-negative RBCs. If laboratories in the UK, Ireland and other European countries validated the use of potent anti-D reagents to result in weak D types 1, 2 and 3 typing initially as RhD-positive, such laboratory results would not require further testing. When serological weak D phenotypes are detected, laboratories should complete RhD testing by determining RHD genotypes (internally or by referral). Individuals with a serological weak D phenotype should be managed as RhD-positive or RhD-negative, according to their RHD genotype.

IgG Subtype Affects Immunomodulation through FcγRs, on Non-Macrophage Populations in a Murine Model - Mechanistic Implications for Anti-D

Background: The administration of anti-D as an immune prophylaxis remains a great success in human immunotherapy; however, the mechanism of anti-D function remains unclear. Currently, anti-D is purified from plasma of D alloimmunized humans; 19 different monoclonal anti-D reagents have been tested but have not shown equivalent efficacy as polyclonal anti-D. While anti-D prevents alloimmunization in most cases, it causes paradoxical enhancement of alloimmunization in certain situations. Likewise, whereas some monoclonal anti-D decrease alloimmunization rates, others enhance. Further progress in refining monoclonals have been limited due to a lack of mechanistic understanding of anti-D.Methods: KEL1 transgenic mice express the human KEL1 antigen selectively on RBCs. Transfusion of KEL1 RBCs into wild-type mice results in alloimmunization to the KEL1 glycoprotein. Polyclonal anti-KEL (from immunized mice) given to naïve mice prevents alloimmunization by KEL1 RBCs. Thus, the KEL1 mouse serves as a murine model of RhD and anti-D [similar to the RhD immune barrier in humans, KEL1 is present on KEL1 mouse RBCs and lacking in wild-type recipients]. We isolated a novel monoclonal anti-KEL1 antibody (PUMA1), sequenced the heavy and light chain variable regions, and generated a panel of recombinant variants of each of the known murine IgG subtypes (IgG1, IgG2a, IgG2b, IgG2c and IgG3) - this gives rise to a panel of antibodies in which IgG subtype is an independent variable, as they each have the same antigen-binding domain. In addition, we generated a novel conditional knockout mouse (Con-FcγR) in which the Fc-receptor common gamma chain (required for expression of all activating murine FcγRs) was deleted upon exposure to CRE recombinase. Con-FcγR mice were crossed with a monocyte/neutrophil specific CRE mouse, resulting in deletion of FcgRs from MØs and neutrophils, but not other tissues.Results: Significant clearance of KEL1+ RBCs was observed with PUMA1 IgG1, IgG2a, and IgG2c, but not IgG2b or IgG3. IgG1 significantly decrease, whereas IgG2a and IgG2c increased anti-KEL alloimmunization (alloimmunization was unaffected by IgG2b or IgG3). When FcγRs were deleted in all tissues, no clearance was observed with any IgG subtype. Likewise, increased anti-KEL alloimmunization by IgG2a and IgG2c was eliminated in FcγR KO mice. In mice with a selective deletion of FcγRs from MØs and neutrophils, clearance of RBCs was eliminated, whereas immunomodulation persisted (decreased with IgG1, increased with IgG2a and IgG2c). Consistent with recent reports in other systems, suppression by IgG1 occurred normally in the FcγKO mice.Conclusions: As in humans, some monoclonal anti-RBC antibodies prevented alloimmunization whereas others enhanced. Inhibiting vs. enhancing RBC alloimmunization was a function of IgG subtype. As both suppressing and enhancing antibodies caused clearance of RBCs, these data dissociate clearance as a mechanistic determinant of suppression vs. enhancement. While clearance and immune enhancement required FcγR dependent pathways, the use of the Con-FcγR mouse demonstrates that while macrophages/neutrophils are required for clearance, macrophages/neutrophils are not required for enhancement of alloimmunization. DisclosuresZimring:Immucor In.: Research Funding; BloodworksNW: Patents & Royalties; Rubius Therapeutics: Membership on an entity’s Board of Directors or advisory committees; State of New Mexico: Consultancy.